brain&transcriptional&profiles&of&male&alternative ... · ! 2! 14!...
TRANSCRIPT
![Page 1: Brain&Transcriptional&Profiles&of&Male&Alternative ... · ! 2! 14! Abstract& 15! ! 16! Bluegill!sunfish!are!one!of!the!classic!systems!for!studying!male!alternative! 17! reproductive!tactics!(ARTs)!in!teleost!fishes.!In!this](https://reader034.vdocuments.mx/reader034/viewer/2022051920/600cc02eb6b92d79104431f5/html5/thumbnails/1.jpg)
! 1!
Brain&Transcriptional&Profiles&of&Male&Alternative&Reproductive&Tactics&in&1!Bluegill&Sunfish&2!&3!Charlyn!G.!Partridge*§,!Matthew!D.!MacManes†,!Rosemary!Knapp¶,!Bryan!D.!Neff*!4!!5!*Department!of!Biology,!University!of!Western!Ontario,!London,!ON,!N6C!1B1!6!Canada!7!†Department!of!Molecular,!Cellular,!and!Biomedical!Sciences,!University!of!New!8!Hampshire,!Durham,!NH!03824,!USA!9!¶Department!of!Biology,!University!of!Oklahoma,!Norman,!OK!73019,!USA!10!§Current!address:!Annis!Water!Resources!Institute,!Grand!Valley!State!University,!11!Muskegon,!MI!49441,!USA!12!! !13!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 2!
Abstract&14!!15!
Bluegill!sunfish!are!one!of!the!classic!systems!for!studying!male!alternative!16!
reproductive!tactics!(ARTs)!in!teleost!fishes.!In!this!species,!there!are!two!distinct!17!
life!histories:!parental!and!cuckolder,!encompassing!three!reproductive!tactics,!18!
parental,!satellite,!and!sneaker.!The!parental!tactic!is!fixed,!whereas!individuals!who!19!
enter!the!cuckolder!life!history!transition!from!the!sneaker!to!the!satellite!tactic!as!20!
they!grow.!For!this!study,!we!used!RNAseq!to!characterize!the!brain!transcriptome!21!
of!the!three!male!tactics!during!spawning!to!identify!gene!categories!associated!with!22!
each!tactic!and!identify!potential!candidate!genes!influencing!their!different!23!
spawning!behaviors.!We!found!that!sneaker!males!had!higher!levels!of!gene!24!
differentiation!compared!to!the!other!two!tactics,!suggesting!that!life!history!does!25!
not!exclusively!drive!differential!gene!expression.!Sneaker!males!had!high!26!
expression!in!ionotropic!glutamate!receptor!genes,!specifically!AMPA!receptors,!27!
which!may!be!important!for!increased!working!spatial!memory!while!attempting!to!28!
cuckold!nests!in!bluegill!colonies.!We!also!found!significant!expression!differences!29!
in!several!candidate!genes!involved!in!ARTs!that!were!previously!identified!in!other!30!
species!and!suggest!a!previously!undescribed!role!for!cytosolic!5’cnucleotidase!II!31!
(nt5c2)!in!influencing!parental!male!behavior!during!spawning.!!!32!
& &33!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 3!
Introduction&34!
Understanding!the!genetic!mechanisms!influencing!variation!in!behavior!can!35!
provide!insight!into!how!different!behavioral!phenotypes!within!populations!evolve!36!
and!are!maintained.!One!important!area!of!research!on!behavioral!phenotypes!37!
focuses!on!alternative!reproductive!tactics!(ARTs),!which!are!found!in!a!wide!array!38!
of!taxa!(Gross!1996;!Mank!&!Avise!2006;!Taborsky!et'al.!2008;!Taborsky!&!39!
Brockman!2010).!ARTs!typically!consist!of!larger!males!practicing!a!“territorial”!40!
tactic!that!maintain!and!protect!breeding!territories!and!smaller!“sneaking”!males!41!
that!sneak!fertilizations!rather!than!compete!with!territorial!males!(Taborsky!42!
1998).!The!mechanisms!underlying!the!expression!of!ARTs!can!differ!significantly!43!
across!species.!In!some!cases,!tactics!are!fixed!for!life!(fixed!tactics)!(Taborsky!44!
1998)!and!often!represent!distinct!life!histories.!Fixed!tactics!can!occur!through!45!
either!inherited!genetic!polymorphisms!(Lank!et'al.!1995;!Shuster!&!Sassaman!46!
1997),!conditioncdependent!switches!that!are!triggered!prior!to!sexual!maturation!47!
(Taborsky!1996;!Gross!1996;!Gross!&!Repka!1998),!or!a!combination!of!genetic!and!48!
environmental!factors!(Piché!et'al.!2008,!Neff!&!Svensson!2013).!In!other!cases,!49!
individuals!can!exhibit!different!tactics!throughout!their!reproductive!life,!either!as!50!
they!grow!or!in!response!to!changing!social!context!(plastic!tactics!or!statusc51!
dependent!tactics)!(Gross!1996;!Taborsky!1996;!Taborsky!et'al.!2008).!Recent!52!
advances!in!genome!sequencing,!such!as!RNA!sequencing!(RNAseq),!now!allow!53!
behavioral!ecologists!to!explore!what!genetic!pathways!contribute!to!behavioral!54!
variation!among!mating!tactics!and!examine!if!the!pathways!differ!among!species!or!55!
between!individuals!that!exhibit!fixed!versus!plastic!tactics.! 56!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 4!
Over!the!past!few!years!there!have!been!numerous!studies!examining!how!57!
differences!in!gene!expression!correlate!with!behaviors!adopted!by!male!ARTs!58!
(AubincHorth!et'al.!2005;'Renn!et'al.!2008;!Fraser!et'al.!2014;!Schunter!et'al.!2014;!59!
Stiver!et'al.!2015).!Most!of!these!studies!have!found!a!large!number!of!genes!within!60!
the!brain!that!vary!in!expression!among!tactics!during!mating.!For!example,!a!recent!61!
study!examining!gene!expression!differences!in!the!ocellated!wrasse!(Symphodus'62!
ocellatus)!found!1,048!differentially!expressed!genes!when!comparing!sneakers!to!63!
two!other!male!tactics!(nesting!and!satellite)!and!to!females!(Stiver!et'al.!2015).!In!64!
the!blackcfaced!blenny!(Tripterygion'delaisi),!RNAseq!identified!approximately!600!65!
transcripts!differentially!expressed!within!the!brains!of!sneaker!versus!territorial!66!
males!(Schunter!et'al.!2014).!In!a!third!study,!approximately!2,000!transcripts!were!67!
differentially!expressed!between!intermediatelycsized!sailfin!molly!(Poecilia'68!
latipinna)!males!that!primarily!perform!courtship!behaviors!compared!to!small!69!
males!that!only!perform!sneaking!behaviors!(Fraser!et'al.!2014).!Changes!in!social!70!
context!also!led!to!a!larger!response!(i.e.!changes!in!gene!expression)!in!71!
intermediatecsized!males!that!show!higher!levels!of!tactic!plasticity!when!compared!72!
to!small!sneaker!males!(Fraser!et'al.!2014),!suggesting!that!genes!driving!neural!73!
response!during!mating!may!differ!between!plastic!and!fixed!tactics.!74!
With!the!increase!in!genomic!studies!examining!differential!gene!expression!75!
among!male!ARTs,!there!are!a!growing!number!of!candidate!genes!suggested!to!76!
drive!the!behavioral!differences!among!tactics.!Schunter!et'al.!(2014)!proposed!a!list!77!
of!potential!candidate!genes!based!on!a!number!of!studies!that!included!78!
gonadotropin!releasing!hormone!(gnrh),!arginine!vasotocin!(avt),!cytochrome!P450!79!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 5!
family!19!subfamily!A!polypeptide!1!(cyp19a1),!ependymin!(epd),!galanin!(gal),!80!
stomatostatin!(sstr1!and!sstr3),!and!early!growth!response!1!(egr1).!Many!of!these!81!
genes!are!involved!in!hormone!regulation!and!mating!behavior,!and!differences!in!82!
expression!levels!have!been!observed!among!mating!tactics!in!different!fish!species!83!
(Table!1).!For!example,!the!product!of!the!cyp19a1b!gene!is!aromatase!B,!a!key!84!
enzyme!responsible!for!the!conversion!of!androgens!to!estrogens!within!radial!glial!85!
cells!of!adult!fish!(Forlano!and!Bass!2005;!Le!Page!et'al.!2010).!Cyp19a1!plays!an!86!
important!role!in!sex!determination!and!sex!change!in!fish!(Nakamura!&!Kobayashi!87!
2005;!Black!et'al.!2005;!Marsh!et'al.!2006)!and!higher!levels!of!gene!expression!have!88!
been!observed!in!territorial!males!compared!to!sneaker!males!in!peacock!blennies!89!
(Salaria'pavo)!(Gonçalves!et'al.!2008),!blackcfaced!blennies!(Tripterygion'delaisi)!90!
(Schunter!et'al.!2015),!and!an!African!cichlid!(Astatotilapia'burtoni)!(Renn!et'al.!91!
2008).!As!more!genomic!data!become!available,!the!number!of!candidate!genes!in!92!
this!list!will!likely!increase!and!evaluating!gene!responses!across!taxa!will!aid!in!93!
determining!whether!similar!genetic!pathways!drive!ART!behaviors!across!different!94!
species.!!95!
One!of!the!bestcstudied!fish!species!with!male!ARTs!is!the!bluegill!sunfish!96!
(Lepomis'macrochirus).!In!this!species,!males!have!two!distinct!life!histories:!97!
parental!and!cuckolder.!In!Lake!Opinicon!(Ontario,!Canada),!parental!males!mature!98!
at!around!seven!years!old!and!construct!nests,!court!females,!and!provide!care!to!99!
young!(Gross!1982).!Cuckolder!males!mature!at!a!significantly!younger!age,!around!100!
two!years!old!(Gross!1982).!Rather!than!competing!with!parental!males!for!access!101!
to!females,!cuckolders!initially!use!a!“sneaking”!tactic!to!dart!in!and!out!of!nests!102!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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while!parental!males!and!females!are!spawning.!As!they!grow,!typically!around!an!103!
age!of!4!years,!cuckolder!males!transition!into!“satellite”!males!by!taking!on!femalec104!
like!coloration!and!behaviors!(Dominey!1980;!Gross!1982).!Satellite!males!use!this!105!
female!mimicry!to!deceive!a!parental!male!that!he!has!two!true!females!in!his!nest!106!
(Neff!&!Gross!2001).!The!parental!and!cuckolder!life!histories!are!fixed!–!once!a!107!
male!adopts!the!parental!or!cuckolder!life!history,!he!remains!in!that!life!history!for!108!
life!(Gross!&!Charnov!1980).!However,!within!the!cuckolder!life!history,!mating!109!
tactics!are!ontologically!plastic,!with!males!apparently!transitioning!from!the!110!
sneaker!tactic!to!the!satellite!tactic!as!they!age!(Gross!&!Charnov!1980).!!111!
While!the!spawning!behavior,!reproductive!success,!and!hormone!profiles!of!112!
bluegill!have!been!studied!extensively!(Gross!&!Charnov!1980;!Kindler!et'al.!1980;!113!
Kindler!et'al.!1991;!Neff!2001;!Neff!2004;!Knapp!&!Neff!2007),!the!genetic!factors!114!
influencing!behavioral!differences!during!spawning!are!less!clear!(Partridge!et'al.!115!
2015).!Thus,!for!this!study,!we!used!RNAseq!to!characterize!the!brain!transcriptome!116!
of!the!three!spawning!male!tactics!(parental,!sneaker,!and!satellite),!in!addition!to!117!
noncspawning!parental!males,!to!examine!how!differences!in!gene!expression!may!118!
relate!to!behavioral!variation!among!the!tactics.!Specifically,!we!(1)!assessed!119!
whether!or!not!there!is!a!greater!difference!in!gene!expression!profiles!between!120!
fixed!tactics!(parental!versus!the!two!cuckolder!tactics)!than!between!tactics!within!121!
a!plastic!life!history!(sneaker!versus!satellite),!(2)!identified!specific!gene!categories!122!
that!are!expressed!for!each!tactic,!and!(3)!examined!the!expression!of!potential!123!
candidate!genes!associated!with!ARTs!from!other!fish!species!to!determine!if!they!124!
also!differentiate!the!ARTs!in!bluegill.!!!125!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 7!
!126!
Methods&and&Materials&127!
Bluegill!Sampling!128!
! In!June!2013,!bluegill!sunfish!were!collected!from!Lake!Opinion!near!Queen’s!129!
University!Biological!Station!(QUBS),!Elgin,!Ontario,!Canada.!A!total!of!12!parental!130!
males,!12!sneaker!males,!13!satellite!males,!and!12!females!were!collected!in!the!act!131!
of!spawning!directly!from!the!bluegill!colony.!An!additional!12!noncnesting!parental!132!
males!were!collected!four!days!prior!to!spawning!(as!determined!once!spawning!at!133!
these!colonies!began).!Individuals!were!euthanized!using!clove!oil,!total!body!length!134!
was!measured,!and!brains!were!immediately!dissected!out!and!stored!in!RNAlater!135!
(Life!Technologies,!Carlsbad,!CA).!Brains!remained!in!RNAlater!at!4°C!for!24!hours!136!
and!were!then!transferred!to!fresh!cryovials,!flash!frozen,!and!kept!in!liquid!137!
nitrogen!until!they!were!transported!on!dry!ice!to!the!University!of!Western!138!
Ontario.!Samples!were!then!stored!at!c80°C!until!RNA!extraction.!!139!
!140!
Total!RNA!Extraction!!141!
! Total!RNA!was!extracted!using!a!standard!Trizol!(Life!Technologies,!142!
Carlsbad,!CA)!extraction.!RNA!was!submitted!to!the!London!Genomics!Center!at!the!143!
University!of!Western!Ontario!and!quality!was!assessed!using!a!2100!Bioanalyzer!144!
(Agilent!Technologies,!Palo!Alto,!CA).!Four!individuals!from!each!group!(spawning!145!
parental!males,!noncspawning!parental!males,!sneaker!males,!satellite!males,!and!146!
females),!for!a!total!of!20!individuals,!were!submitted!to!the!Michigan!State!147!
University!Research!Technology!Support!Facility!c!Genomics!Center!for!cDNA!148!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 8!
Library!Construction!and!Sequencing.!Individuals!used!for!this!study!had!RIN!(RNA!149!
Integrity!Number)!values!ranging!from!9.2c9.9.!150!
!151!
cDNA!Library!Construction!and!Sequencing!152!
! The!cDNA!libraries!were!constructed!for!each!individual!using!Illumina!153!
TrueSeq!Stranded!mRNA!Library!Preparation!Kits!LT!(Illumina,!San!Diego,!CA),!with!154!
each!individual!receiving!a!uniquely!identifiable!index!tag.!The!quality!of!each!155!
library!was!evaluated!and!the!20!individuals!were!multiplexed!into!a!single!sample!156!
that!was!subsequently!run!on!two!lanes!of!an!Illumina!HiSeq2500!Rapid!Run!flow!157!
cell!(v1).!Sequencing!was!performed!on!paired!end!2!x!150!bp!format!reads!and!158!
bases!were!called!using!Illumina!Real!Time!Analysis!software!(v1.17.21.3).!Reads!159!
from!each!individual!were!identified!based!on!their!unique!index!tag,!separated,!and!160!
converted!to!fastq!files!using!Illumina!Bcl2fastq!v1.8.4.!Sequencing!produced!an!161!
average!of!14.5!million!reads!per!individual,!with!over!90%!of!the!reads!having!a!Qc162!
score!>30.!!163!
!164!
De'novo!Transcriptome!Assembly!and!Reference!Transcriptome!165!
! Prior!to!assembly,!read!quality!was!assessed!using!FastQC!166!
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc).!Nucleotides!whose!167!
quality!score!was!below!PHRED=2!were!trimmed!using!Trimmomatic!version!0.32!168!
(Bolger!et'al.!2014),!following!recommendations!from!MacManes!(2014).!The!169!
reference!transcriptome!was!assembled!de'novo!using!Trinity!version!2.04!(Haas!et'170!
al.!2013,!Grabherr!et'al.!2011).!One!representative!of!each!of!the!five!groups!171!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 9!
(spawning!parental!male,!noncspawning!parental!male,!sneaker!male,!satellite!male,!172!
and!female)!was!used!to!construct!a!combined!reference!transcriptome.!The!five!173!
representatives!selected!for!the!reference!were!the!individuals!with!the!highest!174!
number!of!reads!within!their!group!and,!a!total!of!85!million!pairedcend!reads!were!175!
assembled.!The!assembly!was!conducted!with!both!normalized!and!noncnormalized!176!
reads!and!normalization!was!performed!using!Trinity’s!in'silico!normalization!177!
program.!To!test!the!completeness!of!the!transcriptome,!reads!from!samples!not!178!
used!in!the!assembly!were!mapped!back!to!the!transcriptome!using!Burrowsc179!
Wheeler!Aligner!(bwa)cmem!version!0.7.12!(Li!2013),!and!>90%!of!those!reads!180!
aligned,!which!is!comparable!to!the!rate!of!mapping!for!the!individuals!used!in!the!181!
assembly!(92%).!!!182!
TransDecoder!was!used!to!identify!proteinccoding!regions!within!the!183!
assembled!transcriptome.!Transcripts!that!contained!proteinccoding!regions!or!184!
transcripts!that!blasted!to!compete!coding!sequences!(cds)!and!nonccoding!RNA!185!
(ncRNA)!from!Tetraodon'nigroviridis,'Lepisosteus'oculatus,'Xiphophorus'186!
maculatus,'Oryzias'latipes,'Takifugu'rubripes,'Latimeria'chalumnae,'Astyanax'187!
mexicanus,'Danio'rerio,!or!Poecilia'formosa!(downloaded!from!Ensembl)!comprised!188!
the!reference!transcriptome!used!for!both!read!alignment!and!to!estimate!transcript!189!
counts.!!!190!
!191!
Read!Alignment!and!Transcript!Counts!192!
! Reads!from!each!individual!were!separately!aligned!to!the!reference!193!
transcriptome!using!bwacmem!0.7.10!(Li!2013).!At!least!85%!of!all!reads!from!each!194!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 10!
individual!mapped!back!to!the!reference,!with!the!majority!aligning!90%!of!reads!or!195!
higher.!The!sequence!alignment/map!(sam)!files!were!then!converted!to!a!binary!196!
format!(bam)!using!Samtools!0.1.19!(Li!et'al.!2009).!Transcript!counts!for!each!197!
individual!were!obtained!using!the!program!eXpress!1.5.1!(Roberts!&!Pachter!198!
2013).!Differential!gene!expression!was!determined!using!the!R!statistical!package!199!
edgeR!3.6.8!(Robinson!et'al.!2010).!Low!abundance!transcripts!were!filtered!out,!200!
leaving!19,804!transcripts!for!differential!analysis.!Transcript!counts!were!201!
normalized!to!account!for!differences!in!cDNA!library!size!among!individuals!and!202!
dispersion!parameters!were!estimated!using!Tagwise!dispersion!estimates.!!203!
Differences!in!gene!expression!comparing!paired!treatments!were!calculated!using!204!
an!Exactctest!for!binomial!distribution.!Genes!with!pcvalues!lower!than!0.05!after!205!
false!discovery!rate!(FDR)!correction!were!determined!to!be!statistically!significant.!206!
Multidimensional!clustering!analysis!was!used!to!cluster!individuals!together!based!207!
on!the!biological!coefficient!of!variation.!!!208!
!209!
Gene!Annotation!and!Enrichment!Analysis!210!
! Both!the!reference!transcriptome!and!transcripts!differentially!expressed!211!
among!groups!were!blasted!using!Blastx!against!a!customcassembled!fish!protein!212!
database.!This!database!consisted!of!Ensembl!protein!databases!of!13!different!fish!213!
species:!Amazon!molly!(Poecilia'formosa),!zebrafish!(Danio'rerio),!blind!cave!tetra!214!
(Astyanax'mexicanus),!cod!(Gadus'morhua),!coelancanth!(Latimeria'chalumnae),!215!
Japanese!pufferfish!(Takifugu'rubripes),!sea!lamprey!(Petromyzon'marinus),!medaka!216!
(Oryzias'latipes),!platyfish!(Xiphophorus'maculatus),!spotted!gar!(Lepisosteus'217!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 11!
oculatus),!threecspined!stickleback!(Gasterosteus'aculeatus),!greencspotted!218!
pufferfish!(Tetradon'nigroviridis),!and!Nile!tilapia!(Oreochromis'niloticus).!Blast!hits!219!
with!ecvalues!less!than!1x10c10!were!considered!significant.!Ensembl!IDs!from!the!220!
blast!hits!were!then!converted!into!GO!term!identifiers!using!Biology!Database!221!
Network!(bioDBnet)!(http://biodbnet.abcc.ncifcrf.gov/db/dbFind.php).!!222!
For!purposes!of!gene!annotation!and!enrichment!analysis,!we!focused!on!223!
transcripts!within!the!reference!transcriptome!that!were!not!filtered!out!of!the!data!224!
set!due!to!low!transcript!expression!(total!of!19,804!transcripts).!To!examine!which!225!
GO!terms!were!significantly!enriched!within!this!set,!unique!Ensembl!IDs!from!226!
Blastx!were!converted!to!Ensemble!IDs!associated!with!stickleback!homologs!using!227!
the!R!package!biomaRt!2.20.0.!Enrichment!analysis!was!then!conducted!on!the!228!
homologs!using!the!BioMart!portal!(http://central.biomart.org/enrichment).!!229!
For!the!transcripts!that!were!differentially!expressed!among!behavioral!230!
groups,!enrichment!analysis!was!conducted!using!a!Fisher!Exact!test!to!examine!231!
whether!the!proportion!of!genes!within!each!GO!category!was!significantly!higher!232!
than!what!would!be!expected!based!upon!the!proportion!of!genes!assigned!to!that!233!
GO!term!within!the!reference!transcriptome.!To!ensure!adequate!statistical!power,!234!
only!GO!terms!with!at!least!10!transcripts!within!each!category!were!included!in!the!235!
statistical!analysis.!A!FDR!correction!was!applied!to!control!for!multiple!testing!and!236!
GO!terms!with!pcvalues!<!0.05!were!considered!to!be!significant.!!237!
&238!
&239!
&240!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 12!
Results&241!
Reference!Transcriptome&242!
! This!study!presents!the!first!reference!transcriptome!for!the!brain!of!bluegill!243!
sunfish.!The!fully!assembled!transcriptome!consisted!of!272,189!transcripts.!Of!244!
these,!72,189!transcripts!contained!complete!coding!sequences!or!blasted!to!cds!or!245!
ncRNA!from!the!customized!Ensembl!fish!database.!These!72,189!transcripts!were!246!
then!used!as!the!reference!transcriptome!for!alignment!and!mapping.!!The!mean!247!
transcript!length!within!the!reference!transcriptome!was!2,024!bp,!with!N50!=!248!
3,106!bp!and!N90!=!1,018!bp.!The!largest!transcript!consisted!of!27,880!bp.!!249!
Approximately!82%!of!the!transcripts!had!only!one!isoform,!while!18%!(12,951!250!
transcripts)!had!two!or!more!isoforms.!!251!
For!GO!enrichment!analysis,!we!only!examined!the!19,804!transcripts!within!252!
the!reference!transcriptome!that!passed!the!filtering!process.!Of!these,!18,108!had!253!
significant!Blastx!hits!with!Ensembl!gene!IDs!(Table!S1,!Supporting!Information),!of!254!
which!12,224!transcripts!had!stickleback!homologs!that!could!be!used!to!examine!255!
GO!term!enrichment!for!bluegill!sunfish!compared!to!the!stickleback!genome.!The!256!
GO!terms!with!significant!enrichment!included!translation,!catabolism,!vesiclec257!
mediated!transport,!biosynthesis,!small!molecule!metabolism,!and!generation!of!258!
precursor!metabolites!and!energy!(Fig.!1!A!&!B).!!!259!
!260!
Differential!Gene!Expression!among!Groups!261!
Based!on!the!biological!coefficient!of!variation,!sneaker!males!grouped!separately!262!
from!the!other!male!tactics.!However,!noncspawning!parental!males,!spawning!263!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 13!
parental!males,!and!satellite!males!showed!a!large!amount!of!overlap!in!how!264!
transcripts!varied!in!their!expression!levels!(Fig.!2).!The!largest!number!of!265!
differentially!expressed!genes!was!observed!when!comparing!spawning!parental!266!
males!to!sneaker!males,!followed!by!satellite!males!compared!to!sneaker!males,!267!
spawning!parental!males!compared!to!satellite!males,!and!then!spawning!parental!268!
males!compared!to!noncspawning!parental!males!(Table!2).!Analysis!of!sex!269!
differences!in!differential!expression!will!be!presented!in!a!companion!paper!270!
(Partridge!et!al.!in'preparation).!271!
!272!
Differential'Expression'between'Life'Histories'273!
Spawning!Parental!Males!versus!Sneaker!Males.'!A!total!of!9,279!transcripts!were!274!
differentially!expressed!between!parental!males!and!sneaker!males.!Of!these,!4,537!275!
showed!higher!expression!in!parental!males!(Table!S2,!Supporting!Information),!276!
and!4,742!transcripts!showed!higher!expression!in!sneaker!males!(Table!S3,!277!
Supporting!Information).!Enrichment!analysis!of!GO!terms!associated!with!278!
differentially!expressed!genes!showed!that!the!biological!processes!most!enriched!279!
in!parental!males!included!translational!initiation,!translation!elongation,!and!280!
oxidationcreduction!processes!(Table!S4,!Supporting!Information).!The!molecular!281!
function!GO!terms!associated!with!translational!processes!included!structural!282!
constituents!of!the!ribosome!and!translation!initiation!factor!activity!(Table!S4,!283!
Supporting!Information).!Biological!processes!enriched!with!genes!displaying!284!
higher!expression!in!sneaker!males!included!ion!transport,!hemophilic!cell!adhesion!285!
(primarily!due!to!protocadherinc!and!cadherincrelated!genes),!the!ionotropic!286!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 14!
glutamate!receptor!signaling!pathway,!protein!phosphorylation,!and!synaptic!287!
transmission!(Fig.!3A).!Similarly,!significantly!enriched!molecular!function!terms!288!
included!ionotropic!glutamate!receptor!activity!and!protein!binding!(Table!S4,!289!
Supporting!Information).!!290!
!291!
Spawning!Parental!Males!versus!Satellite!Males.!A!total!of!1,141!transcripts!were!292!
differentially!expressed!between!parental!males!and!satellite!males.!Of!these,!676!293!
displayed!higher!expression!in!parental!males!(Table!S5,!Supporting!Information)!294!
and!465!showed!higher!expression!in!satellite!males!(Table!S6,!Supporting!295!
Information).!Only!one!GO!term!related!to!biological!processes!was!enriched!in!296!
parental!males!compared!to!satellite!males!and!this!was!oxidationcreduction!297!
processes.!Significantly!enriched!molecular!functions!included!iron!ion!binding,!298!
oxidoreductase!activity,!and!heme!binding.!Biological!process!terms!enriched!with!299!
genes!showing!higher!expression!in!satellite!males!included!ion!transport,!and!300!
enriched!molecular!function!terms!included!nucleic!acid!binding,!ion!channel!301!
activity,!and!GTP!binding!(Table!S4,!Supporting!Information).!302!
'303!
Differential'Expression'within'Life'Histories'304!
Satellite!Males!verses!Sneaker!Males.!There!were!2,590!transcripts!differentially!305!
expressed!between!satellite!males!and!sneaker!males.!Of!these!transcripts,!1,261!306!
showed!higher!expression!in!satellite!males!(Table!S7,!Supporting!Information),!and!307!
1,329!showed!higher!expression!in!sneaker!males!(Table!S8,!Supporting!308!
Information).!Biological!processes!enriched!with!genes!displaying!higher!expression!309!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 15!
in!satellite!males!included!translation,!embryo!development,!and!cell!cycle!310!
regulation.!Molecular!processes!enriched!in!satellite!males!included!structural!311!
constituents!of!the!ribosome,!heme!binding,!and!oxidoreductase!activity!(Table!S4,!312!
Supporting!Information).!Transcripts!showing!higher!expression!in!sneaker!males!313!
were!involved!in!biological!processes!related!to!ionotropic!glutamate!receptor!314!
signaling!pathways!and!mRNA!processing!(Fig.!3B).!Molecular!functions!were!315!
primarily!related!to!ionotropic!glutamate!receptor!activity!and!protein!binding!316!
(Table!S4,!Supporting!Information).!317!
!318!
Spawning!Parental!Males!verses!NoncSpawning!Parental!Males.!A!total!of!137!319!
transcripts!were!differentially!expressed!between!spawning!and!noncspawning!320!
parental!males.!The!majority!of!these!transcripts!(132!transcripts)!showed!higher!321!
expression!in!spawning!males!(Table!S9,!Supporting!Information).!Genes!with!the!322!
highest!expression!in!spawning!males!compared!to!noncspawning!males!were!MHC!323!
II!antigen!beta!chain,!cytosolic!5’cnucleotidase!II!(nt5c2),!cAMP!responsive!element!324!
modulator!(crem),!cysteine!dioxygenase!type!1!(cdo1),!and!an!uncharacterized!325!
protein.!Only!eight!transcripts!showed!higher!expression!in!noncspawning!parental!326!
males.!These!were!nuclear!receptor!subfamily!1!group!D!member!4b!(nr1d4b),!327!
neuronal!tyrosinecphosphoinositidec3ckinase!adaptor!2!(nyap2),!sphingosinec1c328!
phosphate!receptor!4!(s1pr4),!gammacaminobutyric!acid!A!receptor!beta!3!(gabrb3),!329!
and!four!uncharacterized!proteins.!Due!to!the!limited!number!of!transcripts!330!
differentially!expressed!between!these!two!groups,!the!number!of!transcripts!331!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 16!
assigned!to!each!GO!term!was!too!small!to!have!adequate!statistical!power!to!332!
perform!an!enrichment!analysis!for!this!comparison.!!333!
Potential'Candidate'Genes'Associated'with'ART'Spawning'Behavior'334!
We!observed!differential!expression!in!a!number!of!transcripts!previously!335!
identified!as!potential!candidate!genes!(described!in!Table!1)!associated!with!336!
differences!in!ART!spawning!behaviors!(Table!3).!In!our!data!set,!the!candidate!337!
genes!cytochrome!P450!family!19!subfamily!A!polypeptide!1b!(cyp19a1b),!338!
ependymin!(epd),!and!galanin!(gal)!showed!higher!expression!in!parental!males!339!
compared!to!sneaker!males.!Epd!also!had!higher!expression!in!satellite!males!340!
compared!to!sneakers.!Early!growth!response!1!(egr1)!showed!higher!expression!in!341!
both!satellite!and!sneaker!males!relative!to!spawning!parental!males.!Somatostatin!342!
1!(sstr1)!showed!higher!expression!in!sneaker!males!compared!to!satellite!males,!343!
but!no!differences!in!other!comparisons!between!tactics.!No!differences!in!344!
expression!related!to!gonadotropin!releasing!hormone!(gnrh),!arginine!vasotocin!345!
(avt),!or!somatostatin!3!(sstr3)!were!observed!between!any!of!our!groups.!346!
In!addition!to!these!previously!identified!candidate!genes,!transcripts!that!347!
displayed!some!of!the!highest!differences!in!expression!between!parental!males!and!348!
all!other!male!phenotypes!(including!noncspawning!males)!were!related!to!cytosolic!349!
5’cnucleotidase!II!(nt5c2).!Multiple!isoforms!were!expressed,!with!log2!fold!changes!350!
ranging!from!1.5!–!4.8!times!higher!in!parental!males!compared!to!other!male!351!
groups!(Fig.!4).!Consistent!with!the!finding!for!GO!term!enrichment,!transcripts!that!352!
showed!the!highest!levels!of!expression!in!sneaker!males!compared!to!other!groups!353!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 17!
were!related!to!glutamate!receptor!genes,!particularly!AMPA!ionotropic!glutamate!354!
receptors!(Table!S3,!Supporting!Information).&355!
&356!
Discussion&357!
Bluegill!sunfish!are!a!classic!system!for!examining!behavioral!differences!in!358!
ARTs.!In!this!study,!we!generated!and!assembled!the!first!bluegill!brain!359!
transcriptome,!and!we!identified!candidate!genes!that!contribute!to!differences!in!360!
male!spawning!tactics.!The!main!differences!in!gene!expression!were!found!between!361!
sneaker!males!when!compared!to!the!two!other!male!tactics.!Generally,!sneaker!362!
males!showed!higher!expression!in!transcripts!influencing!neural!activity,!whereas!363!
parental!and!satellite!males!exhibited!higher!expression!in!genes!related!to!364!
translation!and!oxidoreductase!activity.!!365!
One!of!our!key!findings!is!that!a!shared!life!history!does!not!appear!to!be!a!366!
driving!factor!influencing!similarity!in!gene!expression!in!the!brain!of!male!tactics.!367!
In!bluegill,!parental!and!cuckolder!life!histories!are!fixed,!but!within!the!cuckolder!368!
life!history,!males!transition!from!the!sneaking!to!the!satellite!tactic!as!they!age!369!
(Gross!1982;!Gross!&!Charnov!1991).!Our!data!showed!that,!regardless!of!whether!370!
comparisons!were!made!across!fixed!(parental!versus!sneaker!or!parental!versus!371!
satellite)!or!plastic!(sneaker!versus!satellite)!tactics,!sneaker!males!showed!the!372!
highest!level!of!differentiation!in!gene!transcription.!Similar!results!have!been!373!
observed!in!the!ocellated!wrasse,!Symphodus'ocellatus,!where!sneaker!males!also!374!
showed!the!greatest!number!of!differentially!expressed!genes!compared!to!nesting!375!
and!satellite!males!(Stiver!et'al.!2015).!The!expression!differences!in!sneakers!376!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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! 18!
compared!to!the!other!tactics!in!bluegill!and!the!ocellated!wrasse!are!likely!partially!377!
due!to!age!because!sneaker!males!are!typically!younger!than!satellite!and!parental!378!
or!territorial!males.!Indeed,!a!recent!study!in!the!shortclived!fish!Nothobranchius'379!
furzeri!found!that!genes!related!to!translation!elongation!and!ribosomal!proteins!are!380!
upcregulated!with!age!(Baumgart!et'al.!2014).!Both!translation!elongation!and!381!
ribosomal!proteins!showed!higher!expression!in!parental!and!satellite!males!382!
compared!to!sneaker!males!in!our!data!set.!Additionally,!the!behaviors!exhibited!by!383!
sneaker!males!during!spawning!differ!in!fundamental!aspects!from!those!of!the!384!
other!male!tactics.!In!the!ocellated!wrasse,!for!example,!satellite!and!nesting!males!385!
cooperatively!protect!the!nest!from!sneakers!and!other!egg!predators!(Taborsky!et'386!
al.!1987);!in!bluegill,!satellite!and!parental!males!associate!closely!with!the!female!387!
throughout!spawning,!whereas!sneakers!dart!in!and!out!of!the!nest.!These!388!
differences!in!spawning!tactics!likely!also!contribute!to!the!differences!in!gene!389!
expression!observed!in!the!two!studies.!!Thus,!age!and!spawning!tactic!are!390!
important!contributors!to!gene!expression!patterns!across!ARTS,!and!life!history!is!391!
not!exclusively!responsible!for!these!differences.!!392!
!Identifying!distinct!gene!categories!expressed!by!one!ART!type!compared!to!393!
another!provides!information!regarding!the!genes!influencing!behavioral!394!
differences!during!spawning.!Previous!studies!in!sailfin!mollies,!Poecilia'latipinna,!395!
and!Atlantic!salmon,!Salmo'salar,!indicate!that!sneaker!males!have!increased!396!
expression!in!genes!related!to!neurotransmission!and!learning!(AubincHorth!et'al.!397!
2005;!Fraser!et'al.!2014).!We!found!that!the!GO!terms!consistently!enriched!in!398!
bluegill!sneaker!males!compared!to!both!parental!and!satellite!males!were!the!399!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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ionotropic!glutamate!signaling!pathway!and!ionotropic!gluatamate!receptor!activity.!400!
Ionotropic!gluatamate!receptors!are!primarily!excitatory!neurotransmitter!401!
receptors!and!play!an!important!role!in!fast!synaptic!transmission!(reviewed!in!402!
Lamprecht!&!LeDoux!2004).!Two!of!these!receptors,!NMDA!and!AMPA,!play!403!
important!roles!in!memory!function!and!spatial!learning!(reviewed!in!Riedel!et'al.!404!
2003).!Blocking!NMDA!receptors!impairs!learning!new!spatial!locations!in!goldfish!405!
(Gómez!et'al.!2006).!Futhermore,!mice!with!impaired!AMPA!receptors,!while!406!
showing!normal!spatial!learning,!have!impaired!working!spatial!memory!(i.e.!their!407!
ability!to!alter!their!spatial!choice!in!response!to!changing!environments!is!408!
impaired)!(Reisel!et'al.!2002).!We!propose!that!increased!expression!of!genes!409!
related!to!spatial!memory,!particularly!related!to!working!spatial!memory,!could!be!410!
important!for!bluegill!sneakers!during!spawning!as!they!attempt!to!gain!access!to!411!
nests!while!avoiding!detection!not!only!by!the!parental!males,!but!also!predators!412!
that!are!common!around!the!colony!(Gross!&!MacMillian!1981).!Bluegill!sneakers!413!
must!also!position!themselves!in!close!proximity!to!females!so!they!can!time!sperm!414!
release!to!coincide!with!female!egg!release!(Stoltz!&!Neff!2006).!Similarly,!sailfin!415!
molly!sneakers,!who!also!show!enrichment!in!ionotropic!glutamate!related!genes!416!
(Fraser!et'al.!2014),!probably!benefit!from!increased!working!spatial!memory!417!
because!they!must!successfully!position!themselves!by!the!female!for!quick!and!418!
successful!copulations.!In!this!context,!increased!expression!in!gene!pathways!that!419!
improve!neural!function!related!to!working!spatial!memory!are!probably!especially!420!
beneficial!to!the!sneaker!tactic!to!increase!their!reproductive!success.!!421!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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There!are!a!number!of!candidate!genes!that!have!been!proposed!to!drive!the!422!
expression!of!alternative!mating!tactics!(Schunter!et'al.!2015).!In!our!study!of!423!
bluegill,!we!corroborate!some!of!these!candidates.!For!example,!cyp19a1b,!epd,!and!424!
gal!had!higher!expression!levels!in!parental!males!compared!to!sneaker!males.!The!425!
expression!patterns!for!all!three!genes!are!similar!to!what!has!been!observed!in!426!
cichlids!(Renn!et'al.!2008).!In!addition,!expression!of!epd!is!lower!in!rainbow!trout,!427!
Oncorhynchus'mykiss,!males!that!use!a!sneaking!tactic!versus!males!that!are!428!
dominant!and!territorial!(Sneedon!et'al.!2011),!which!is!also!consistent!with!our!429!
findings.!In!contrast,!the!one!candidate!gene!that!responded!opposite!to!430!
expectations!was!egr1.!Egr1'expression!was!lower!in!bluegill!parental!males!431!
compared!to!sneaker!or!satellite!males!although!previous!work!on!cichlids!found!432!
that!expression!of!this!gene!increases!when!subdominant!males!transition!into!433!
dominant!males!(Burmeister!et'al.!2005).!However,!egr1!is!an!important!434!
transcription!factor!involved!in!neural!plasticity!(Jones!et'al.!2001),!so!it!may!be!435!
involved!in!regulating!the!switch!from!one!tactic!to!another.!Consequently,!in!436!
bluegill,!this!gene!would!be!more!important!for!individuals!that!alter!their!tactic!437!
(sneaker!to!satellite)!than!for!the!fixed!parental!tactic.!Taken!together,!our!results!438!
corroborate!a!role!for!cyp19a1b,!epd,'gal,'and'egr1'as!candidate!genes!contributing!439!
to!behavioral!differences!in!ARTs!across!species.!!440!
We!also!found!a!transcript!that!may!have!a!previously!unrecognized!function!441!
in!influencing!male!spawning!behavior.!Transcripts!corresponding!to!splice!variants!442!
of!cytosolic!5’cnucleotidase!II!(nt5c2)!were!significantly!higher!in!parental!males!443!
when!compared!to!all!other!male!groups,!including!noncspawning!males.!The!444!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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protein!product!of!nt5c2!regulates!purine!metabolism!(Bretonnet!et'al.!2005;!445!
Walldén!et'al.!2007).!Moreover,!in!the!African!cichlid,!A.'burtoni,!uridine!kinase!446!
(udk)!expression,!a!gene!with!similar!function!to'nt5c2,!is!significantly!higher!in!447!
dominant!relative!to!subordinate!males!(Renn!et'al.!2008).!While!neither!nt5c2'nor!448!
udk'have!been!directly!associated!with!spawning!behavior!in!fishes,!there!is!449!
evidence!suggesting!that!altered!expression!levels!of!nt5c2!in!the!brain!can!450!
significantly!influence!anxiety,!mania,!schizophrenia,!and!aggressive!behaviors!in!451!
humans!(Page!et'al.!2007),!and!altering!levels!of!uridine!in!mice!affects!their!level!of!452!
aggression!toward!an!intruder!(Kawasaki!et'al.!2013).!Furthermore,!in!bluegill,!453!
parental!males!display!high!levels!of!aggression!to!obtain!nesting!sites,!circumvent!454!
cuckoldry,!and!prevent!egg!predation!by!brood!predators!(Avila!1976;!Colgan!et'al.!455!
1979;!Gross!1979;!Gross!&!Macmillian!1981).!The!high!levels!of!nt5c2'expression!in!456!
spawning!parental!males!suggests!that!this!gene'may!have!a!role!in!influencing!457!
parental!male!spawning!behaviors!in!bluegill.!Future!work!should!examine!how!458!
nt5c2!influences!mating!behaviors!in!the!ARTs!of!this!and!other!species.!!459!
In!summary,!our!work!describes!differences!in!gene!expression!profiles!in!460!
the!brains!of!bluegill!male!ARTs!during!spawning.!The!largest!differences!in!461!
expression!levels!were!observed!when!comparing!sneakers!to!parental!and!satellite!462!
males,!suggesting!that,!in!bluegill,!tactic!is!more!related!to!differences!in!gene!463!
expression!than!is!life!history.!Consistent!with!other!studies,!our!work!demonstrates!464!
that!sneaker!males!have!greater!expression!of!genes!involved!in!neural!function!465!
relative!to!more!territorialctype!males,!particularly!in!relation!to!working!spatial!466!
memory,!as!mediated!by!ionotropic!glutamate!receptors.!We!found!support!for!the!467!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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previously!identified!candidate!genes!cyp19a1b,!epd,'gal,'and'egr1!contributing!to!468!
behavioral!differences!in!ARTs,!but!we!also!show!evidence!for!a!novel!candidate!469!
gene,!nt5c2,!implicated!in!these!differences.!We!suggest!that!nt5c2'may!have!a'role!470!
in!mediating!courtship!or!territorial!behaviors!within!this!species,!and!we!471!
recommend!that!future!work!should!characterize!this!gene!further!in!other!species.!472!
!473!
Acknowledgments&474!
We!thank!Scott!Colborne!for!his!help!in!collecting!bluegill,!Dave!Bridges!for!475!
providing!the!R!script!to!convert!Ensemble!IDs!to!stickleback!homologs,!and!David!476!
Winter!and!Jeramia!Ory!for!providing!Python!script!used!in!the!bioinformatics!477!
analyses.!We!also!thank!Shawn!Garner,!Tim!Hain,!Lauren!Kordonowy,!and!Lindsay!478!
Havens!for!helpful!comments!on!the!manuscript.!Funding!for!this!project!was!479!
supported!through!the!Natural!Sciences!and!Engineering!Research!Council!of!480!
Canada!(NSERC)!to!B.D.N.!and!the!University!of!Oklahoma!Department!of!Biology!to!481!
R.K.!!482!
!483!
!484!
485!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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increased!cellular!nucleotidase!activity.!Proceedings'of'the'National'Academy'656!of'Sciences'USA,!94,!11601c11606.!657!
!658!Piché!J,!Hutchings!JA,!Blanchard!W!(2008)!Genetic!variation!in!threshold!reaction!659!
norms!for!alternative!reproductive!tactics!in!Atlantic!salmon,!Salmo'salar.!!660!Proceedings'of'the'Royal'Society'B:'Biological'Sciences,!275,!1571c1575.!!661!
!662!Reisel!D,!Bannerman!DM,!Schmitt!WB,!Deacon!RMJ,!Flit!J,!Borchardt!T,!Seeburg!PH,!663!
Rawlins!JNP!(2002)!Spatial!memory!dissociations!in!mice!lacking!GluR1.!664!Nature'Neuroscience,!5,!868c873.!!!665!
!666!Renn!SC,!AubincHorth!N,!Hofmann!HA!(2008)!Fish!and!chips:!functional!genomics!of!667!
social!plasticity!in!an!African!cichlid!fish.!Journal'of'Experimental'Biology,!668!211,!3041c3056. 669!
for this preprint is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder; http://dx.doi.org/10.1101/025916doi: bioRxiv preprint first posted online September 2, 2015;
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670!Riedel!G,!Platt!B,!Micheau!J!(2003)!Glutamate!receptor!function!in!learning!and!671!
memory.!Behavioural'Brain'Research,!140,!1c47.!!672!!673!Roberts!A,!!Pachter!L!(2013)!Streaming!fragment!assignment!for!realctime!analysis!674!
of!sequencing!experiments.!Nature'Methods,!10,!1c47.!675!!676!Robinson!MD,!McCarthy!DJ,!Smyth!GK!(2010)!EdgeR:!a!Bioconductor!package!for!677!
differential!expression!analysis!of!digital!gene!expression!data.!678!Bioinformatics,!26,!139c140.!679!
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and!differential!gene!expression!in!a!noncmodel!fish!species!with!alternative!682!mating!tactics.!BMC'Genomics,!15,!167.!!683!
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and!sex!ratio!in!a!marine!isopod.!Nature,!388,!373c377.!!686!!687!Sneddon!LU,!Schmidt!R,!Fang!Y,!Cossins!AR!(2011)!Molecular!correlates!of!social!688!
dominance:!A!novel!role!for!ependymin!in!aggression.!PLoS'One.!6,!e18181.!!689!!690!Stiver!KA,!Harris!RM,!Townsend!JP,!Hofmann!HA,!Alonzo!SH!(2015)!Neural!gene!691!
expression!profiles!and!androgen!levels!underlie!alternative!reproductive!692!tactics!in!the!ocellated!wrasse,!Symphodus'ocellatus.!Ethology,!121,!152c167.!693!
!694!Stoltz!JA,!Neff!BD!(2006)!Male!size!and!mating!tactic!influence!proximity!to!females!695!
during!sperm!competition!in!bluegill!sunfish.!Behavioral'Ecology'and'696!Sociobiology,!59,!811c818.!697!
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phenotypes.!In:!Animal'Behavior:'Evolution'and'Mechanisms!(Ed.!P.!Kappeler).!703!Springer,!Berlin,!537c586.!704!
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Symphodus!(Crenilabrus)!ocellatus,!a!European!wrasse!with!four!types!of!707!male!behaviour.!Behaviour,!102,!82c117.!708!
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reproductive!tactics:!concepts!and!questions.!In:!Alternative'Reproductive'711!Tactics:'An'Integrative'Approach!(Eds.!Oliveira!RF,!Taborksy!M,!Brockmann!712!HJ)!Cambridge!University!Press,!1c21.!713!
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Thomson!JS,!Watts!PC,!Pottinger!TG,!Sneddon,!LU!(2011)!Physiological!and!genetic!715!correlates!of!boldness:!Characterizing!the!mechanisms!of!behavioral!716!variation!in!rainbow!trout,!Oncorhynchus'mykiss.!Hormones'and'Behavior,!59,!717!67c74.!718!
!719!Walldén!K,!Stenmark!P,!Nyman!T,!Flodin!S,!Gräslund!S,!Loppnau!P,!Norlund!P!720!
(2007).!Crystal!structure!of!human!cytosolic!5’cnucleotidase!II.!Insights!into!721!allosteric!regulation!and!substrate!recognition.!Journal'of'Biological'722!Chemistry,!282,!17828c17836.!723!
!724!Wu!Z,!Autry!AE,!Bergan!JF,!WatabecUchida!M,!Dulac!CG!(2014)!Galanin!neurons!in!725!
the!medial!preoptic!area!govern!parental!behavior.!Nature,!509,!325c330.!!726! 727!!728!
Author&Contribution&729!
C.G.P,!R.K.,!and!B.D.N.!designed!the!experiment!and!collected!samples.!M.D.M.!730!
assembled!the!transcriptome.!C.G.P.!performed!bioinformatic!and!statistical!731!
analyses!and!wrote!the!manuscript.!All!authors!provided!comments,!contributed!to!732!
the!manuscript,!and!approved!the!final!manuscript.!!733!
!734!
Data&Accessibility&735!
All!raw!sequence!files!are!available!on!the!Sequence!Read!Archive!(SRA)!736!
through!BioProject!ID:!PRJNA287763.!Environmental!data,!RNA!quality!information,!737!
assembled!transcriptome,!the!transcript!count!matrix,!and!R!code!for!differential!738!
gene!analysis!are!available!on!Dryad!(http://dx.doi.org/10.5061/dryad.82fd8!and!739!
http://dx.doi.org/10.5061/dryad.10hh7).!!!740!
!741!
Supporting&Information&742!
Table!S1:!Annotated!Reference!Transcriptome!743!!744!Table!S2:!Transcripts!with!significantly!higher!expression!in!bluegill!parental!males!745!compared!to!sneaker!males.!!746!
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!747!Table!S3:!Transcripts!with!significantly!higher!expression!in!bluegill!sneaker!males!748!compared!to!parental!males.!749!!750!Table!S4:!Biological!Process!and!Molecular!Function!GO!terms!that!are!significantly!751!enriched!with!genes!differentially!expressed!between!tactics!752!!753!Table!S5:!Transcripts!with!significantly!higher!expression!in!bluegill!parental!males!754!compared!to!satellite!males.!!755!!756!Table!S6:!Transcripts!with!significantly!higher!expression!in!bluegill!satellite!males!757!compared!to!parental!males.!!758!!759!Table!S7:!Transcripts!with!significantly!higher!expression!in!bluegill!satellite!males!760!compared!to!sneaker!males.!!761!!762!Table!S8:!Transcripts!with!significantly!higher!expression!in!bluegill!sneaker!males!763!compared!to!satellite!males.!!764!!765!Table!S9:!Transcripts!with!significantly!higher!expression!in!spawning!parental!766!males!compared!to!noncspawning!parental!males.!!767!
&768!
! !769!
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Table!1:!Proposed!candidate!genes!(Schunter!et'al.!2014)!influencing!teleost!alternative!reproductive!770!tactics!(ARTs).!POA!=!Precoptic!area!771!
&&772!&773!!774!!775!!776!! !777!
Proposed(Candidate(Genes Function Relationship(to(ARTsArginine'Vasotocin'(avt) Non1mammalian'homolog'of'vasopressin.'
Activates'some'aspects'of'sexual'behavior!'in'posterior'POA'of'territorial'cichlid'males,'but'!'anterior'POA'of'non1territorial''(Greenwood$et$al.'2008);'"'density'of'avt'mRNA'in'POA'in'parental'blenny'males'(Grober'et$al.$2002)
Gonadotrophin'Releasing'Hormone'(gnrh) Regulates'release'of'lutenizing'hormone'and'follicle1stimulating'hormone'from'the'pituitary'gland'
!'in'territorial'cichlid'males'(Renn'et$al.'2008)
Cytochrome'P450'family'19,'subfamily'A,'polypeptide'1'(cyp19a1)
Brain'aromatase.'Key'enzyme'in'estrogen'biosynthesis
!'in'territorial'cichlid'males'(Renn'et$al.'2008);!'territorial'blenny'males'(Gonçalves'et$al.'2008);'!'territorial'black1faced'blenny'males'(Schunter'et$al.'2014);'"'in'the'sonic'motor'nucleus'of'nesting'type'1'(territorial)'male'plainfin'midshipmen'compared'to'type'II'(female'mimic)'males'(Forlano'et$al.$2005)
Ependymin'(epd) Glycoprotein'associated'with'neuroplasticity'and'neuronal'regeneration.'Also'affects'aggression'levels'in'zebrafish'(Sneddon'et$al'2011);'associated'with'stress'in'trout'(Thomson'et$al.$2011)
!'in'territorial'cichlid'males'(Renn'et$al.'2008);'"'in'subordinate'trout'males'(Sneddon'et$al.'2011)
Galanin/GMAP'prepropeptide''(gal) Neuropeptide'that'influences'neurotransmitters.'Associated'with'sexual'behaviors'(Bloch'et$al.'1993),'and'parental'care'(Wu'et$al.'2014)
!'in'territorial'cichlid'males'(Renn'et$al.'2008)
Somatostatin'(sst) Neuropeptide'that'regulates'endocrine'pathways.'Also'affects'neurotransmitters'
!'in'territorial'blenny'males'(Schunter'et$al.'2014);'!in'territorial'cichlid'males'(Renn'et$al.'2008)
Early'growth'response'1'(egr1) Transcripton'factor'that'influences'neural'plasticity
!(when'subdominant'cichlid'males'switch'to'dominant'(Burmeister'et$al.'2005)
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Table!2:!Number!of!transcripts!differentially!expressed!(DE)!between!each!male!bluegill!tactic,!778!including!the!number!of!transcripts!without!Blastx!hits,!the!number!with!unique!Ensembl!IDs,!and!779!the!number!of!transcripts!assigned!to!specific!GO!terms!780!
!! !781!
ComparisonsNum,of,DE,Transcripts
Num,of,DE,Transcripts,without,Blastx,Hits
Num,with,Unique,Ensembl,Gene,IDs
Num,DE,Gene,IDs,,with,GO,Annotation
Parental(vs(Sneaker 9,279 516 5,396 2,430Parental(vs(Satellite 1,141 82 879 317Satellite(vs(Sneaker 2,590 184 1,852 351Parental(vs(Non;Spawner( 140 6 102 70
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Table!3:!Gene!expression!differences!(Log2!fold!change)!among!male!tactics!for!proposed!candidate!782!genes!(see!Table!1).!!783!
&&784!Values!in!brackets!represent!pcvalues!after!false!discovery!rate!correction.!Values!in!bold!are!785!significant!at!p!<!0.05.!!Parent!=!parental!male,!Sneak!=!sneaker!male,!Sat!=!satellite!male.!786!&787!! !788!
Proposed(Candidate(Genes Isoform(ID Parent(vs(Sneak Parent(vs(Sat Sat(vs(SneakNon7Spawn(vs(Spawn(Parent(
Arginine'Vasotocin'(avt) c34708_g2_i1 0.45'[0.32] <0.98'[0.09] 0.54'[0.33] <0.74'[0.5]
Gonadotrophin'Releasing'Hormone'(gnrh) 63124_g1_i1 0.76'[0.5] 0.32'[0.87] 0.44'[0.77] <0.77'[0.88]
Cytochrome'P450'19a'1b'(cyp19a1b) c48084_g2_i1 0.93([0.0002] 0.64'[0.06] 0.28'[0.4] <0.39'[0.58]
Ependymin'(epd) c44195_g1_i5 1.54([1.4(x(1078] 0.66'[0.07] 0.89([0.007] 0.51'[0.45]
Galanin/GMAP'prepropeptide''(gal) c41071_g5_i2 1.12([0.0001] 0.53'[0.91] <0.59'[0.1] <0.09'[0.97]
Somatostatin'1'(sstr1) c3001_g1_i1 0.53'[0.15] <0.39'[0.49] 0.93([0.03] <0.27'[0.88]
Somatostatin'3'(sstr3) c46547_g6_i1 0.001'[1] <0.25'[0.54] 0.25'[0.48] <0.15'[0.9]
Early'growth'response'1'(egr1) c37907_g1_i1 70.74([0.02] 70.91([0.03] 0.16'[0.72] 0.63'[0.42]
Comparison(between(Male(Tactics(((Log2(Fold(Change)(
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!789!Fig.!1:!GO!terms!related!(A)!biological!processes!and!(B)!molecular!function!that!790!were!significantly!enriched!in!the!bluegill!reference!transcriptome!relative!to!the!791!stickleback!genome.!Boxes!of!similar!color!can!be!grouped!into!the!same!GO!term!792!hierarchy.!The!size!of!each!box!reflects!the!–log10!pcvalue!of!the!GO!term.!793!!794!!795!!796!!797!!798!!799!!800!!801!!802!!803!!804!!805!!806!!807!!808!!809!!810!!811!!812!
B) Enriched Molecular Functions
enzyme regulator activity
GTPase activitykinase activity
RNA binding
structural constituent of ribosome
unfolded proteinbinding
A) Enriched Biological Processes
mRNA processing
nucleobase-containing compound catabolic process
protein folding
translation
nucleo-cytoplasmic
transport tran
spor
t
vesicle-mediated transport
catabolic process
bios
ynth
etic
pr
oces
s small molecule metabolic process
generation of precursor metabolites and energy
Reference Transcriptome
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!813!!814!Fig.!2:!Multicdimensional!space!(MDS)!plot!based!on!the!biological!coefficient!of!815!variation!(bcv)!among!bluegill!male!ARTs.!Red!triangles:!sneaker!males,!green!816!pluses:!satellite!males,!black!circles:!spawning!parental!males,!blue!x:!noncspawning!817!parental!males.!818!!819!!820!!821!!822!!823!! !824!
−0.4 −0.2 0.0 0.2 0.4 0.6
−0.4
0.0
0.2
0.4
bcv1
bcv2
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!825!!826!Figure!3:!GO!terms!significantly!enriched!by!genes!with!higher!expression!in!827!sneaker!males!compared!to!(A)!parental!males!and!(B)!satellite!males.!Boxes!of!828!similar!color!are!grouped!into!the!same!GO!term!hierarchy.!Box!size!reflects!the!–829!log10!pcvalue!of!the!GO!term.!830!!831!!832!!833!!834!!835!!836!!837!!838!!839!!840!!841!!842!!843!!844!!845!!846!!847!!848!
A) Parentals vs Sneakers – higher in sneakers
peptidyl-tyrosine dephosphorylation
protein phosphorylation
RN
A-de
pend
ent
DN
Are
plic
atio
n
cell adhesion
homophilic cell adhesion via
plasma membrane adhesion molecules
calcium ion transmembrane
transport
ion transport
potassium ion
transport
regulationof ion
transmembranetransport
transport
ionotropic glutamatereceptor signaling pathway
pyrimidine nucleobase
catabolic process
regulation of ARF GTPase
activity
regulation of transcription,
DNA-templated
signal transduction
synaptic transmission
microtubule-based
movementcell
communication
B) Satellites vs Sneakers – higher in sneakers
mR
NA
proc
essi
ng
ion transporttransmembrane
transport
transport
ionotropicglutamate receptorsignaling pathway
synaptictransmission
termination ofG-protein coupled
receptorsignalingpathway
RNA-
depe
nden
tDN
Are
plic
atio
n
regulationof ion
transmembranetransport
calcium ion trans-
membrane transport
potassium ion
transport
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!849!Figure!4:!Log2!fold!changes!of!sneaker,!satellite,!and!noncspawning!(NS)!parental!850!males!relative!to!spawning!parental!males!for!cytosolic!5’cnucleotidase!II!(nt5c2).!*!851!indicates!fold!changes!that!are!significantly!different!with!pcvalues!<!0.05!after!FDR!852!correction.!!853!!854!
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